RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium

Abstract

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.

FIG. 1.

Venn diagram of all significant (B > 0) gene expression changes within the comparison of L354 (S. Typhimurium SL1344) against L133 (S. Typhimurium ramA::aph), L1007 (S. Typhimurium ramR::aph), and L786 (S. Typhimurium pTRChisA-ramA). Selected genes from each strain are displayed. Boldface shows increased gene expression; lightface shows decreased gene expression.

FIG. 2.

RT-PCR for six SPI genes for ramA::aph (A), pTRChisA-ramA (B), and ramR::aph (C) (asterisks show significant [P < 0.05] differences) and RT-PCR for acrB, tolC, and ramA (D) (all RT-PCR data shown are significant; P < 0.05). Black bars, microarray data; gray bars, RT-PCR data; dashed line, fold change of 1 (i.e., no change); for all array changes, a B value of >0 is considered significant.

FIG. 3.

Western blotting for SipC after growth of strains SL1344, L133, and L786 in minimal medium, in the presence and absence of IPTG (to induce overexpression of ramA) and ampicillin (to ensure maintenance of the pTRC plasmid).

FIG. 4.

Adhesion to (A) and survival in (B) RAW 264.7 mouse macrophages of SL1344, L133, and L786 (with IPTG).

FIG. 5.

Colonization of BALB/c mice by S. Typhimurium SL1344 and L133 (ramA::aph). Mice were inoculated orally with 4 × 10⁷ CFU (A) or intravenously with 2.4 × 10³ CFU (B). The CFU per organ of SL1344 (circles) and L133 (squares) and the geometric mean (horizontal bar) are indicated. P values were calculated using the Mann-Whitney test.

FIG. 6.

Survival of Caenorhabditis elegans after infection with SL1344 (diamonds), L133 (squares), L1007 (circles), or L786 (triangles).